The broad, long-term objective of this research program is to develop general technology to conditionally regulate protein function at the level of the protein molecules rather than by targeting the DNA or mRNA precursors that encode a protein-of-interest. This technology is highly specific for the targeted protein and provides rapid and tunable control of protein function using cell-permeable small molecules. The goal is to engineer small protein domains called destabilizing domains that are rapidly and conditionally degraded when expressed in mammalian cells. The instability of a destabilizing domain is faithfully conferred to other proteins fused to these small domains. A cell-permeable ligand that binds tightly to the destabilizing domains regulates their stability. The genetic fusion of the destabilizing domain to the gene-of-interest ensures specificity, and the attendant small-molecule control confers speed, reversibility and dose-dependence to this method. The reagents developed to date involve domains that are unstable in the absence of ligand and stabilized when ligand binds. The specific aims of this proposal are designed to deliver complementary new domains that are destabilized rather than stabilized by the addition of the cell-permeable ligand. A mechanistic understanding of how these domains are recognized and degraded in mammalian cells will make this technology more useful to users. These studies may also reveal general mechanisms that cells use to recognize and degrade unfolded or misfolded proteins, and these mechanisms are likely relevant to important human diseases. PUBLIC HEALTH RELEVANCE: The goal of this research program is to develop novel methods to rapidly and reversibly regulate the stability of specific proteins in eukaryotic cells and living mammals. This methodology would enable the development of many new models for human diseases, and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.